Summary/Abstract:Forhumanstoproperlydevelop,thefertilizedeggmustdivideina mannerthateventuallyresultsinabodythathasallofthecorrectorgansandappendagesinall ofthecorrectplaces.Muchofthisisdeterminedbythepropertemporalandspatialregulation ofmasterregulatorygenesthatguideformationofthedifferentiatedcellsthatmakeuporgans andappendages.Akeyaspectofthisdevelopmentalprocessistheabilitytomaintainmaster regulatorygenesinarepressedstateincellswheretheirexpressionmightcauseinappropriate cellbehavior.Itisknownthatmisexpressionofevenasinglemasterregulatorygene,suchas thoseencodedintheHOXloci,canresultinacellbehavinginamannerincompatiblewithits bodylocationandtissuetype.ThePolycomb-??Group(PcG)groupofgenesislargelyresponsible formaintainingthisrepression,baseduponintensivestudyofthisgroupoverthepast70years. Thisapplicationdescribesthecontinuationofourworkonunderstandingthefunctionofthe proteinsencodedbythePcGgenes.Theseproteinsformseveralcomplexes.Thefocusofthis applicationisPolycombRepressiveComplex1(PRC1),themain?engine?ofrepressioninthe Polycomb-??Group.PRC1isknowntointeractwithchromatin,thestructurethatpackagesgenes inthenucleusofcells.ChromatinstructurecanrendertheenclosedDNAinaccessibleto activatingfactors.Aprominenthypothesisinthefieldhasbeenthatrepressioncanbe generatedbygeneratinghighlypackagedDNAthatisnolongerabletobetranscribedand expressed.WewillinvestigatetherolethatPRC1hasingeneratingpackagedchromatininthe firsttwoAims.Wewillinvestigatehowlongnon-??codingRNAsinterfacewithPRC1toregulate itsfunctioninthethirdAim.Specifically,inAim1wewillstudytheknownabilityofPRC1to interactwithnucleosomes,theprimarybuildingblockofchromatin,toformthemintoa compactedstructure.Wewilltestthehypothesisthatcompactionisessentialtofunctionby analyzingthedevelopmentalphenotypes,inmice,ofmutationsthatimpedetheabilityofPRC1 toformcompactedstructures.InAim2wewillinvestigatetheabilityofPRC1togenerate higher-??orderstructures(onthescaleof100kbofDNA)thatarecompactedasvisualizedby superresolutionmicroscopyandbychromatinconformationcapture.Aim3characterizesthe abilityofPRC1complexestobindtolncRNAinvitro.Wewillalsodeveloptechnologiestomap thelocationoflncRNAbindingtochromatinincells.Wewillthencomparetheimpactof specificmutationsinlncRNAsandinPRC1onfunctiontotestthehypothesisthatspecific interactionsbetweenPRC1andlncRNAsareneededforproperfunctionofthePcGsystem.